Optimizing a Difficult Coupling

If you observed very low conversions to your desired product with significant amounts of unreacted starting material, we recommend trying to optimize the reaction by screening a variety of different coupling reagents. Yields may also be improved through repeated coupling reactions, double or triple coupling, or prolonged coupling reactions, overnight, for example.

When optimizing your reaction, please be mindful that activated amino acids can be prone to racemization. Check for literature precedence on specific coupling reagents before attempting to heat your reaction during your optimization, or see “How to check for Epimerization” under “Troubleshooting,” below, to do this yourself.

If you don’t know where to start, the following coupling reagents could be a good starting point: COMU, triphosgene, use caution, Ghosez’ reagent, EEDQ at 60 ˚C. These have shown utility in the synthesis of azapeptides, N-amino peptides, and oligo(N-substituted alanines) for coupling to the unnatural amino acids.

For more information on coupling reagents, read Peptide Coupling Reagents, More than a Letter Soup.

If you are working with difficult peptide sequences that may be prone to aggregation, a different optimization might be necessary. The article The Road to the Synthesis of ‘Difficult Peptides,’ is helpful.

Heating a Reaction on Solid Phase

The heating of a reaction on solid phase can be accomplished a few different ways. Simply placing an SPPS cartridge or microwave vial in an oil bath may work to an extent, but will not allow for agitation of the resin during the reaction. For alternative, likely more efficient procedures, consider the following options:

• Placing your SPPS cartridge in a heated sonicator. Some models allow you to heat between 30-80 ˚C, other models just have an on/off switch for heating and will reach ~ 60 ˚C. If you are near or above the boiling point of your solvent, use microwave vials instead of SPPS cartridges to prevent the caps from popping off your cartridge due to pressure buildup.

• Using jacketed peptide vessels, which are available on chemglass. When connected to a heated water bath circulator, these allow you to circulate warm water around a fritted glass vessel that contains your resin. The resin can undergo mixing by bubbling of nitrogen.

• Microwave Heating

Optimizing your Cleavage Cocktail

You might notice some undesired side reactions during resin cleavage, which can sometimes be prevented by adjusting your cleavage cocktail composition. An excellent quick guide on how to choose a TFA-based cleavage cocktail can be found here.

Dissolving “Tricky Peptides”

As “tricky peptides” we consider insoluble, hydrophobic, and aggregation prone, after ether precipitation for purification.

• If you observe an immiscible phase/oil drop appearance when attempting to dissolve crude peptide, it may result from residual organic solvent, diethyl ether, used for precipitation. Freezing and lyophilizing can help remove residual solvent. We’ve often observed that powders, observed upon lyophilization, are more readily produced after ether precipitation.

• Some peptides require long durations of intense agitation for solvation. Consider exposing peptides immersed in water/MeCN mixtures to sonication at an elevated temperature, up to 50 ˚C for up to 30 minutes. Ultrasonic water bath incubators work well for this purpose.

• Some peptides that fail to dissolve well in standard solvent mixtures, water or 50/50 water/acetonitrile, will dissolve in pure water-miscible organic solvents, DMSO/DMF. We have found frequent success in lyophilizing insoluble crude peptides by first suspending crude peptide in a water/acetonitrile mixture followed by dropwise addition of DMSO until the peptide dissolves. Lyophilization of the resulting solution provides a white powder. If the addition of DMSO/DMF is not acceptable, you can also try resuspending crude peptides first in pure acetonitrile followed by addition of water.

• Peptides which are particularly insoluble may not be amenable to solubilization using any of the methods above. In such cases, you can first dissolve in a small volume of trifluoroacetic acid followed by dilution with MeCN. Such aggressive solvents can prevent the peptide from adhering to reverse phase stationary phase upon injection onto HPLC. Such preparations should be tested on analytical scale to ensure peptide adheres prior to large scale injections.

Avoiding Complications from UV Active Scavengers in Cleavage Cocktails

Additives to cleavage cocktails such as phenol and thioanisole are difficult to remove completely with ether precipitation steps and often show large peaks in HPLC chromatograms. These contaminants can be particularly problematic if they co-elute with desired products. We have found that redesigning the acetonitrile gradient in HPLC, making a steeper or more shallow ascent in acetonitrile concentration, can often provide resolution between scavenger peaks and peptide peaks – even if the two overlap using other gradients.

Purifying hydrophobic peptides on reverse-phase HPLC

Very hydrophobic peptides have been known to irreversibly adhere to reverse-phase columns. Certain tricks can be used to reduce adherence of such hydrophobic peptides. Columns can be heated, to reduce adherence and provide sharper HPLC peaks, using water jackets. Water and acetonitrile HPLC solvents can be supplemented with organic cosolvents, such as isopropanol, although this can increase solvent backpressure due to viscosity changes.